Colchicine-skeleton compounds, their use as medicaments and compositions containing them

ABSTRACT

The present invention relates to colchicine and thiocolchicine derivatives which can be obtained from these molecules by functionalization of the C-7 to ketone or functionalization of the amino group. Said compounds have a marked antiblastic activity both on the normal cancer cells and on the chemoresistant phenotype. The compounds of the invention can be administered both by injection and orally.

This application is a 371 of PCT/EP97/02577, filed May 21, 1997.

TECHNOLOGICAL BACKGROUND

The present invention relates to novel colchicine derivatives havingantiproliferative, antineoplastic and antiinflammatory activities, themethods for the preparation thereof and the pharmaceutical formulationscontaining them.

Colchicine is a known pseudo-alkaloid widely used for a very long timein therapy for the treatment of gout, a pathology on which it acts veryquickly and specifically, even though it should be used for short timesdue to its toxicity. A colchicine derivative, namely thiocolchicoside,is widely used to treat contractures and in inflammatory conditions onskeletal muscles. In addition, colchicine is a very potent antiblasticagent, which acts blocking the formation of the mitotic spindle duringcell division; this latter aspect has been investigated thoroughly forany antineoplastic activity and a great deal of colchicine derivativeshave been prepared to this purpose. Colchicine as such and a number ofits derivatives could not be used clinically due to their high toxicity,and therefore their unacceptable risk/benefit ratio. Only one colchicinederivative, demecoicine, is used in some degree in oncology for thetreatment of some leukemia forms.

Therefore the problem exist of the availability of antineoplasticmedicaments having a satisfactory risk/benefit ratio, i.e. a hightherapeutical activity with poor or no side-effects.

Another problem in the antineoplastic field is the resistance to themedicament which takes place in specific phenotypes.

Now it has surprisingly been found that some colchicine derivatives havea high cytotoxic activity both on the normal cancerous cells and on thecorresponding resistant phenotype (MDR).

The compounds of the invention are potent apoptosis inducers, proving tobe markedly better than the compounds of the prior art. Due to theirlipophilic, characteristics, the compounds are particularly bioavailableafter oral administration. Moreover, the compounds of the presentinvention can be administered by the parenteral or topical routes aswell.

SUMMARY OF THE INVENTION

The present invention relates to compounds of formula (I) ##STR1##wherein R is a methoxy or methylthio group and R₁ is a straight orbranched alkyl or alkenyl group having 1 to 6 carbon atoms, or analicyclic or heterocyclic moiety, a saturated or unsaturated mono ordicarboxylic or amino acidic acyl residue or a β-D-glucose or6-deoxygalactose residue.

Examples of alkyl group are methyl, ethyl, propyl, isopropyl, n-butyl,iso-butyl, t-butyl, pentyl, neopentyl, hexyl.

Examples of alkenyl group are propenyl, 1-butenyl, 2-butenyl,1-pentenyl.

Examples of alicyclic group are cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl.

Examples of heterocyclic group are benzotriazolyl, methyltetrazolyl.

Examples of acyl residue are ximenoyl, succinyl, aspartyl.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of formula I are prepared starting from the naturalcompounds colchicine or thiocolchicine or from the C3-derivativesthereof commercially available or obtainable with methods known inliterature. As described in literature, the C3 derivatives can beprepared by reacting the 3-O-dimethyl derivative with an alkyl or acylhalide. The hydrolysis of said compounds with strong mineral acidaqueous solutions allows to obtain selectively, changing the temperatureand the reaction time, the corresponding N-deacetyl derivatives. Inparticular, the deacetylation of thiocolchicine or of the C3 derivativesthereof can be carried out by subjecting the compounds to acidichydrolysis; in the case of thiocolchicine, the hydrolysis with haloacids or, more preferably, with sulfuric acid (20% H₂ SO₄ --120 h),allows one to obtain N-deacetylthiocolchicine and3-demethyl-N-deacetylthiocolchicine in nearly quantitative yields.

The N-deacetyl derivatives are reacted with4-formyl-1-methylpyridinium-p-toluenesulfonate and1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to prepare the compounds offormula I.

Alternatively, reacting the N-deacetyl derivatives with2,3-ditert-butyl-1,2-benzoquinone, the compounds of formula II areobtained: ##STR2## wherein R and R₁ have the meanings described above.

A further embodiment of the present invention encompasses the compoundsof formula II.

The compounds of the invention exhibit a remarkable antineoplasticactivity both in vitro and in vivo.

The table shows the antimitotic activity of the compounds of theinvention on cultured breast tumour explants normal (MCF-7) or resistantto both adriamycin and vinblastin (MCF7-ADR), compared with colchicineand taxol.

                  TABLE                                                           ______________________________________                                                  IC.sub.50 (nM)                                                      Compounds   MCF7-ADR  MCF-7     MCF7-ADR/MCF7                                 ______________________________________                                        Colchicine  12,000    1.8       6,600                                         Compound Ia 15        6.2       2.4                                           Compound Ib 40        23        1.7                                           Compound IIa                                                                              52        17        3.0                                           Taxol       2,400     2.3       1,043                                         ______________________________________                                    

This table evidences that the compounds of the invention havesignificant advantages on the resistant cell lines, which are nowadaysconsidered the main target of cytotoxic medicaments.

Moreover, the compounds according to the present invention haveantiinflammatory and antiarthritis activities (degenerative rheumatoidarthritis and similar pathologies) and they can be incorporated inpharmaceutical formulations useful for the administration of themedicament for the indicated pathology. Formulations for intravenous,oral, transdermal, epicutaneous administrations can conveniently beprepared.

Among the excipients useful to prepare said formulations, natural andsynthetic phospholipids proved to be particularly useful for preparingliposomial forms for the parenteral and/or topical routes. The sameformulations proved to be useful in the topical treatment of cutaneousepitheliomas and in cutaneous hyperproliferative conditions, such aspsoriasis. In the specific antineoplastic field, besides thephospholipids which allow the administration of the medicament in theliposomial form, some surfactants such as polyethoxylated castor oils orpolysorbates acting synergistically with the active ingredient, turnedout to be particularly useful. Preferably the active principle ismicronized to dissolve the compound in water. A surprisingly active,convenient form is the complex of these compounds with cyclodextrins.

In oncology, the products are used at dosages from 1 to 100 mg/m².

EXAMPLES

The following examples further illustrate the invention.

Example 1

Preparation of Thiocolchicone from N-deacetylthiocolchicine

(Ia: R=SMe; R₁ =Me)

100 ml of CH₂ Cl₂ and 30 ml of DMF are mixed under nitrogen atmosphere,then 4 g of deacetylthiocolchicine (M.W. 373, 10.7 mmol) and 24.2 g of4-formyl-1-methylpyridinium p-toluenesulfonate (M.W. 279, 15 mmol) areadded; the whole is refluxed for 3 hours or until the amine disappears.The solution is cooled to 0° C. and then added with 1.94 g of DBU (M.W.152, 12.8 mmol), drop by drop, to obtain a dark red solution. After 15minutes, 150 ml of an oxalic acid aqueous solution are added, themixture is left to react overnight, then repeatedly extracted with CH₂Cl₂ ; dried over sodium sulfate and the solvent is evaporated off todryness. The residue is crystallized from ethyl acetate to obtain a 78%yield. Thiocolchicone has the following chemical-physical andspectroscopical characteristics.

M.p. 212° C.

MS (E.I.): 372 m/z (35%)-344 (55%)-329 (6%)-311 (13%)-301 (4%)-287(8%)-267 (4%)-243 (4%)-215 (4%)-84 (64%)-49 (100%). ¹ H-NMR (300 MHz,CDCl₃)

    ______________________________________                                        ppm     molt    int     type  J(Hz)  J(Hz)                                                                              J(Hz)                               ______________________________________                                        2.45    s       3H      SME                                                   2.68    ddd     1H      H-5eq.                                                                              13.4   4.8  1.9                                 2.81    ddd     1H      H-6ax 16.6   13.4 4.8                                 2.95    ddd     1H      H-6eq 16.6   5.5  1.9                                 3.11    ddd=td  1H      H-5ax 13.1   13.5 5.6                                 3.57    s       3H      OMe                                                   3.88    s       3H      OMe                                                   3.90    s       3H      OMe                                                   6.56    s       1H      H-4                                                   6.96    s       1H      H-8                                                   7.07    AB      1H      H-11  10.2                                            7.27    AB      1H      H-12  10.2                                            ______________________________________                                    

¹³ C-NMR (300 MHz, CDCl₃): 15.8 ppm (SMe)-30.0 (C-5)-48.0 (C-6)-56.7(OMe-3)-61.8 (OMe-2)-61.8 (OMe-1)-107.8 (C-4)-125.3 (C-12)-127.0(C-11)-130.7 (C-8)-134.4 (C-4a)-136.1 (C-12a)-136.5 (C-12)-142.3(C-2)-150.4 (C-1)-152.6 (C-7a)-154.6 (C-3)-160.7 (C-10)-182.9(C-9)-206.2 (C-7).

Example 2

Preparation of Colchicone from N-deacetyl-colchicine

(Ib: R=OMe; R₁ =Me)

3.58 g of N-deacetylcolchicine are treated according to the procedure ofExample 1. 2.6 g of colchicone are obtained, having the followingchemical-physical and spectroscopical characteristics.

MS (E.I.): 356 m/z (100%)-328 (95%)-313 (25%)-300 (22%)-285 (18%)-271(26%)-253 (13%)-238 (8%)-227 (13%)-199 (16%)-171 (11%). ¹ H-NMR (300MHz, CDCl₃).

    ______________________________________                                        ppm     molt   int     type  J(Hz)  J(Hz)                                                                              J(Hz)                                ______________________________________                                        2.67    ddd    1H      H-5β                                                                           13.7   5.0  2.2                                  2.82    ddd    1H      H-6β                                                                           16.6   13.6 5.0                                  2.95    ddd    1H      H-6α                                                                          16.6   5.4  2.2                                  3.11    ddd    1H      H-5α                                                                          13.7   13.6 5.4                                  3.55    s      3H      OMe-1                                                  3.86    s      3H      OMe-2                                                  3.87    s      3H      OMe-3                                                  4.00    s      3H      OMe-10                                                 6.54    s      1H      H-4                                                    6.85    d      1H      H-11  10.7                                             7.12    s      1H      H-8                                                    7.24    d      1H      H-12  10.7                                             ______________________________________                                    

¹³ C-NMR (300 MHz, CDCl₃): 29.27 ppm (C-5)-43.33 (C-6)-55.96(OMe-10)-56.44 (OMe-3)-61.07 (OMe-2)-61.12 (OMe-1)-106.96 (C-4)-112.40(C-11)-124.50 (C-12b)-132.00 (C-4a)-132.80 (C-8)-135.30 (C-12)-136.15(C-12a)-141.80 (C-2)-150.16 (C-1)-151.83 (C-7a)-153.70 (C-3)-164.10(C-10)-179.40 (C-9)-205.60 (C-7).

Example 3

Preparation of the Condensation Product between Thiocolchicine and3,5-ditert-butyl-1,2-benzoquinone

(IIa: R=SMe; R₁ =Me)

500 mg of deacetylthiocolchicine (M.W. 373, 1.34 mmol) and 590 mg of3,5-di-tert-butyl-1,2-benzoquinone (M.W. 220, 2.69 mmol) are dissolvedin 50 ml of methanol, under normal atmosphere.

The reaction is followed by TLC (CH₂ Cl₂ :acetone 30:1) and after about18 hours the solvent is evaporated off under vacuum.

The warm crude is dissolved in 1 volume of ethyl acetate, 1-1.5 volumesof hexane are added and the mixture is cooled on ice. The reactionproduct is recovered by filtration, the yield being 70%. This compoundhas the following chemical-physical and spectroscopical characteristics.

M.p. 238° C. with decomposition MS (E.I.): 573 m/z (33%)-558 (1%)-545(100%)-530 (9%)-514 (7%)-314 (4%)-301 (4%)-265 (7%)-249 (7%). ¹ H-NMR(300 MHz, CDCl₃).

    ______________________________________                                        ppm      molt   int       type   J(Hz)                                                                              J(Hz)                                   ______________________________________                                        1.30     s      9H        tBu                                                 1.40     s      9H        tBu                                                 2.45     s      3H        SMe                                                 3.05     dd     1H        H-5    14.0 4.0                                     3.30     dd     1H        H-5    14.0 4.0                                     3.55     s      3H        OMe                                                 3.83     s      3H        OMe                                                 3.87     s      3H        OMe                                                 3.86     t      1H        H-6    4.0                                          6.65     s      1H        H-4                                                 7.08     d      1H        H-11   11.0                                         7.20     d      1H        H-5'   2.0                                          7.26     d      1H        H-3'   2.0                                          7.28     d      1H        H-12   11.0                                         7.33     s      1H        H-8                                                 ______________________________________                                    

¹³ C-NMR (300 MHz, CDCl₃): 15.71 ppm (SMe)-30.33 (C(CH₃)₃)-31.95(C(CH₃)₃)-34.96 ((C(CH₃)₃)-35.25 (C(CH₃)₃)-36.60 (C-5)-56.41(OMe-3)-61.81 (OMe-1)-61.64 (OMe-2)-76.47 (C-6)-109.80 (C-4)-123.56(C-5')-124.61 (C-3')-125.75 (C-12b)-126.80 (C-11)-132.28 (C-4')-133.58(C-6')-135.13 (C-8)-135.47 (C-4a)-136.10 (C-12)-137.01-(C-12a)-142.35(C-2)-143.21 (C-7a)-144.88 (C-2')-147.19 (C-1')-152.06 (C-1)-153.78(C-3)-159.85 (C-10)-164.68 (C-7)-182.26 (C-9).

Example 4

    ______________________________________                                        Preparation of tablets containing compound (Ia)                               ______________________________________                                        Compound Ia                25 mg                                              Lactose                    47 mg                                              Microcrystalline cellulose 20 mg                                              Cross-linked sodium carboxymethyl cellulose                                                              5 mg                                               Colloidal silica           1 mg                                               Talc                       1 mg                                               Magnesium stearate         1 mg                                               ______________________________________                                    

Example 5

    ______________________________________                                        Preparation of a liposome cream containing compound                           (IIa)                                                                         ______________________________________                                        Compound IIa            0.20   g                                              Phosphatidylcholine     20.00  g                                              Cholesterol             0.50   g                                              Butylhydroxytoluene     0.01   g                                              95% Ethanol             8.00   g                                              Disodium edetate        0.15   g                                              Imidazolidinyl urea     0.30   g                                              Sodium dehydroacetate   0.20   g                                              Hydroxyethyl cellulose  2.00   g                                              (Natrosol 250 HHX-Aqualon)                                                    Distilled water         67.75                                                 ______________________________________                                    

Example 6

    ______________________________________                                        Preparation of an injectable solution containing                              compound (Ia)                                                                 ______________________________________                                        Compound Ia             15     mg                                             PEG-660 12-hydroxystearate                                                                            2.500  mg                                             Propylene glycol        1.000  mg                                             alcohol q.s. to         5      ml                                             ______________________________________                                    

What is claimed is:
 1. A compound of formula I: ##STR3## wherein R ismethoxy or methylthio; and R₁ is a straight chain or branched alkylgroup having 1 to 6 carbon atoms, a straight chain or branched alkenylgroup having 1 to 6 carbon atoms, a cyclic aliphatic moiety, aheterocyclic moiety, a saturated or unsaturated monocarboxylic acylresidue, a saturated or unsaturated dicarboxylic acyl residue, asaturated or unsaturated amino acid acyl residue, a β-D-glucose residue,or a 6-deoxylgalactose residue, with the proviso that, when R ismethoxy, R₁ is other than methyl or acetyl.
 2. The compound of claim 1wherein R is methoxy.
 3. The compound of claim 1 wherein R ismethylthio.
 4. The compound of claim 3 wherein R₁ is methyl.
 5. Thecompound of formula II: ##STR4## wherein R is methoxy or methylthio; andR₁ is a straight chain or branched alkyl group having 1 to 6 carbonatoms, a straight chain or branched alkenyl group having 1 to 6 carbonatoms, a cyclic aliphatic moiety, a heterocyclic moiety, a saturated orunsaturated monocarboxylic acyl residue, a saturated or unsaturateddicarboxylic acyl residue, a saturated or unsaturated amino acid acylresidue, a β-D-glucose residue, or a 6-deoxylgalactose residue.
 6. Thecompound of claim 5 wherein R is methoxy.
 7. The compound of claim 5wherein R is methylthio.
 8. The compound of claim 6 wherein R₁ ismethyl.
 9. The compound of claim 7 wherein R₁ is methyl.
 10. Apharmaceutical composition suitable for administration to a patientcomprising a pharmaceutically effective amount of the compound of claim1 and an excipient.
 11. A pharmaceutical composition suitable foradministration to a patient comprising a pharmaceutically effectiveamount of the compound of claim 5 and an excipient.
 12. A method ofpreparing a compound of formula I: ##STR5## wherein R is methoxy ormethylthio; and R₁ is a straight chain or branched alkyl group having 1to 6 carbon atoms, a straight chain or branched alkenyl group having 1to 6 carbon atoms, a cyclic aliphatic moiety, a heterocyclic moiety, asaturated or unsaturated monocarboxylic acyl residue, a saturated orunsaturated dicarboxylic acyl residue, a saturated or unsaturated aminoacid acyl residue, a β-D-glucose residue, or a 6-deoxylgalactoseresidue, with the proviso that, when R is methoxy, R₁ is other thanmethyl or acetyl; comprising:selecting a first compound from the groupconsisting of N-deacetylcolchicine and N-deacetylthiocolchicine; andreacting said first compound with 4-formyl-1-methylpyridiniump-toluenesulfonate and 1,8-diazabicyclo[5.4.0]undec-7-ene at atemperature and for a time sufficient to form the compound of formula I.13. A method of preparing a compound of formula II: ##STR6## wherein Ris methoxy or methylthio; and R₁ is a straight chain or branched alkylgroup having 1 to 6 carbon atoms, a straight chain or branched alkenylgroup having 1 to 6 carbon atoms, a cyclic aliphatic moiety, aheterocyclic moiety, a saturated or unsaturated monocarboxylic acylresidue, a saturated or unsaturated dicarboxylic acyl residue, asaturated or unsaturated amino acid acyl residue, a β-D-glucose residue,or a 6-deoxylgalactose residue; comprising:selecting a first compoundfrom the group consisting of N-deacetylcolchicine andN-deacetylthiocolchicine; and reacting said first compound with3,5-ditert-buty-1,2-benzoquinone at a temperature and for a timesufficient to form the compound of formula II.